The analysis of macromolecular or particle species in solution is usually achieved by preparing a sample in an appropriate solvent and then injecting an aliquot thereof into a separation system such as a liquid chromatography (LC) column or Field Flow Fractionation (FFF) channel wherein the different species of particles contained within the sample are separated into their various constituencies. Once separated by such means, generally based on size, mass, or column affinity, the samples are subjected to analysis by means of light scattering, refractive index, UV absorption, electrophoretic mobility, viscometric response, etc. In this disclosure we will primarily concern ourselves with multi-angle light scattering (MALS).
A typical HPLC-MALS setup is shown in FIG. 2. Solvent is generally drawn by an HPLC pump 201 from a solvent reservoir 202 through a degasser 203 and then pumped through filtering means 204 to the injection valve 211. A liquid sample 205 is injected into the sample loop 212 of the injection valve 211, generally by a syringe 206. The sample, however, may be added to the flow stream by means of an auto injector rather than the manual means described. The fluid sample then flows from the injector through one or more HPLC columns 207, where the molecules or particles contained within the sample are separated by size, with the largest particles eluting first. The separated sample then passes sequentially through a MALS detector 208 and a concentration detector such as a differential refractometer 209 before passing to waste. Other instruments capable of measuring the physical properties of the molecules or particles in the sample may also be present along the flow stream. For example, a UV/Vis absorbance detector and/or a viscometer might be present within the chain of instruments. In general, data generated by the instruments is transmitted to a computer that is capable of collecting, storing, and analyzing the data and reporting the results to the user.
As discussed above, a sample containing aliquot is injected into a separation system, such as the HPLC system shown in FIG. 2, or, as relates more closely to the present invention, a UHPLC system which is similar but wherein the columns 207 are one or more UHPLC columns rather than HPLC columns, and the pump 201 is capable of producing the higher pressures at which UHPLC systems operate. The columns separate the sample by size into its constituent fractions. Each of these eluting and separated fractions results in a “peak” which passes via tubing to a measurement volume. Each detection instrument measures, in turn, a signal from the peak as it passes through the measurement volume. A single aliquot may generate any number of peaks, for example, a monodisperse sample of single particle size will generate only one peak. As each sample peak passes through a measurement volume a signal will be detected that relates to the sample being analyzed at any given instant as it passes through the cell. These finite measurements are often referred to as “slices,” each slice representing the instantaneous measurement of the sample being detected at a given volume of eluent flowing through the cell. These signals are digitized and stored in a computer, and the resultant data is generally reported to the user as a peak 301, such as that shown in FIG. 3, wherein each element of the peak represents a given slice. The data at a given slice, in this case the light scattering data over a plurality of angles, 302, corresponding to a single slice 302a may be viewed and analyzed utilizing a software program such as ASTRA®. The data shown in FIG. 3 also includes the signal 303 recorded from a differential refractometer.